The chloroplasts of higher plants contain many unique, interconnected biochemical pathways that produce an array of secondary metabolite compounds which not only perform vital functions within the plant but are also important from agricultural and nutritional perspectives. Three such secondary metabolites are the lipid soluble, chloroplastically synthesized compounds vitamin E (.alpha.-tocopherol or .alpha.-toc), plastoquinones (PQ), and carotenoids, which together perform many crucial biochemical functions in the chloroplast. PQ and vitamin E are quinone compounds synthesized by a common pathway in the plastid; carotenoids are tetraterpenoids synthesized by a separate plastid-localized pathway.
Plastoquinone (PQ) often accounts for up to 50% of the total plastidic quinone pool in green tissues. The primary function of PQ is as a fundamental component of the photosynthetic electron transport chain, acting as an electron carrier between photosystem II and the cytochrome b.sub.6 f complex. PQ likely has other less well studied functions in plastids, namely in acting as a direct or intermediate electron carrier for a variety of other biosynthetic reactions in the chloroplast.
Vitamin E is the second major class of chloroplastic quinones, accounting for up to 40% of the quinone pool in plastids. The essential nutritional value of tocopherols was recognized around 1925, and the compound responsible for Vitamin E activity was first identified as .alpha.-tocopherol in 1936. .alpha.-Toc has a well-documented role in mammals as an antioxidant, and a similar, though less well understood antioxidant role in plants. Liebler, et al., Toxicology 23:147-169, 1993; Hess, Anti-oxidants in Higher Plants, CRC Press: 111-134, 1993.
Carotenoids are a separate, diverse group of lipophilic pigments synthesized in plants, fungi, and bacteria. In photosynthetic tissues, carotenoids function as accessory pigments in light harvesting and play important roles in photo-protection by quenching free radicals, singlet oxygen, and other reactive species. Siefermann-Harms, Physiol. Plantarum. 69:561-568, 1987. In the plastids of non-photosynthetic tissues, high levels of carotenoids often accumulate providing the intense orange, yellow, and red coloration of many fruits, vegetables, and flowers (Pfander, Methods in Enzym., 213A, 3-13, 1992). In addition to their many functions in plants, carotenoids and their metabolites also have important functions in animals, where they serve as the major source of Vitamin A (retinol), and have been identified as providing protection from some forms of cancer due to their antioxidant activities. Vitamin E's antioxidant activities are also thought to protect against some forms of cancer, and may act synergistically with carotenoids in this regard. Liebler, et al., Toxicology 23:147-169, 1993; Krinsky, J. Nutr. 119:123-126, 1989.
Tocopherol and Plastoquinone Synthesis
.alpha.-Tocopherol and plastoquinone are the most abundant quinones in the plastid and are synthesized by the common pathway shown in FIG. 1. The precursor molecule for both compounds, homogentisic acid (HGA), is produced in the chloroplast from the shikimic acid pathway intermediate p-hydroxyphenyl pyruvic acid (pOHPP), in an oxidation/decarboxylation reaction catalyzed by the enzyme p-hydroxyphenyl pyruvic acid dioxygenase (pOHPP dioxygenase). Homogentisic acid is subject to phytylation/prenylation (phytylpyrophosphate and solanylpyrophosphate, C.sub.20 and C.sub.45, respectively) coupled to a simultaneous decarboxylation by a phytyl/prenyl transferase to form the first true tocopherol and plastoquinone intermediates, 2-demethylphytylplastoquinol and 2-demethylplastoquinol-9, respectively. A single ring methylation occurs on 2-demethylplastoquinol to yield plastoquinol-9 (PQH.sub.2) which is then oxidized to plastoquinone-9 (PQ). This oxidation is reversible and is the basis of electron transport by plastoquinone in the chloroplast.
The preferred route, as established in spinach, for .alpha.-tocopherol formation from 2-demethylphytylplastoquinol appears to be 1) ring methylation of the intermediate, 2-.alpha.-demethylphytylplastoquinol, to yield phytylplastoquinol, 2) cyclization to yield d-tocopherol and, finally, 3) a second ring methylation to yield .alpha.-tocopherol. Ring methylation in both tocopherol and plastoquinone synthesis is carried out by a single enzyme that is specific for the site of methylation on the ring, but has relatively broad substrate specificity and accommodates both classes of quinone compounds. This methylation enzyme is the only enzyme of the pathway that has been purified from plants to date. d'Harlingue, et al., J.Biol.Chem. 26:15200, 1985. All enzymatic activities of the .alpha.-toc/PQ pathway have been localized to the inner chloroplast envelope by cell fractionation studies except for pOHPP dioxygenase and the tocopherol cyclase enzyme. Difficulties with cell fractionation methods, low activities for some of the enzymes, substrate stability and availability and assay problems, make studying the pathway biochemically difficult.
Vitamin E and PQ levels, ratios, and total amounts vary by orders of magnitude in different plants, tissues and developmental stages. Such variations indicate that the vitamin E and PQ pathway is both highly regulated and has the potential for manipulation to modify the absolute levels and ratios of the two end products. The pathway in FIG. 1 makes it clear that production of homogentisic acid by pOHPP dioxygenase is likely to be a key regulatory point for bulk flow through the pathway, both because HGA production is the first committed step in .alpha.-toc/PQ synthesis, and also because the reaction is essentially irreversible. Therefore modifying the levels of HGA by modifying pOHPP dioxygenase activity should have a direct impact on the total .alpha.-toc/PQ biosynthetic accumulation in plant tissues, and, as described below, because of the connection of PQ and carotenoid synthesis, should also affect carotenoid synthesis in plant tissues.
Carotenoid Biosynthesis; Quinones as Electron Carriers
In plants, carotenoids are synthesized and accumulate exclusively in plastids via the pathway shown on the left-hand side of FIG. 1. The first committed step in carotenoid synthesis is the condensation of two molecules of the C.sub.20 hydrocarbon geranylgeranyl pyrophosphate (GGDP) by the enzyme phytoene synthase, to form the colorless C.sub.40 hydrocarbon, phytoene. In oxygenic photosynthetic organisms (e.g. plants, algae, and cyanobacteria), phytoene undergoes two sequential desaturation reactions, catalyzed by phytoene desaturase, to produce .zeta.-carotene through the intermediate phytofluene. Subsequently, .zeta.-carotene undergoes two further desaturations, catalyzed by .zeta.-carotene desaturase, to yield the red pigment lycopene. Lycopene is cyclized to produce either .alpha.-carotene or .beta.-carotene, both of which are subject to various hydroxylation and epoxidation reactions to yield the carotenoids and xanthophylls most abundant in photosynthetic tissues of plants, lutein, .beta.-carotene, violaxanthin and neoxanthin.
The genes encoding the first two enzymes of the carotenoid pathway (phytoene synthase and phytoene desaturase) have been isolated and studied from a number of plant and bacterial sources in recent years. Sandmann, Eur. J. Biochem. 223:7-24, 1994. Phytoene desaturase has been the most intensively studied, both because it is a target for numerous commercially important herbicides, and also because the phytoene desaturation reaction is thought to be a rate limiting step in carotenoid synthesis. Molecular and biochemical studies suggest that two types of phytoene desaturase enzymes have evolved by independent evolution: the crtI-type found in anoxygenic photosynthetic organisms (e.g. Rhodobacter and Erwinia), and the pds-type found in oxygenic photosynthetic organisms. Despite their differences in primary amino acid sequence, all phytoene desaturase enzymes contain a dinucleotide binding domain (FAD or NAD/NADP), which in Capsicum annum has been shown to be FAD. Hugueney et al., Eur. J. Biochem. 209:399-407, 1992. Presumably, the bound dinucleotide in both types of phytoene desaturase enzymes is reduced during desaturation and reoxidized by an unknown reductant present in the plastid or bacterium.
Several lines of evidence have suggested a role for quinones in the phytoene desaturation reaction in higher plants. Using isolated daffodil chromoplasts, Mayer and co-workers demonstrated that in an anaerobic environment, oxidized artificial quinones were required for the desaturation of phytoene while reduced quinones were ineffective. Mayer et al., Eur. J. Biochem. 191:359-363, 1990. Further supporting evidence comes from studies with the triketone class of herbicides (e.g. Sulcotrione), which cause phytoene accumulation in treated tissues but unlike the well-studied pyridazone class (e.g. Norflorazon (NFZ)) do not directly affect the phytoene desaturase enzyme. Rather, triketone herbicides competitively inhibit pOHPP dioxygenase, an enzyme common to the synthesis of both plastoquinone and tocopherols, suggesting that one or more classes of quinones may play a role in carotenoid desaturation reactions. Schulz et al., FEBS 318:162-166, 1993; Secor, Plant Physiol. 106: 1429-1433; Beyer et al., IUPAC Pure and Applied Chemistry 66:1047-1056, 1994.
Despite the well-studied, wide-spread importance of vitamin E, plastoquinone, and carotenoids to human nutrition, agriculture, and biochemical processes within plant cells, much remains unclear about their biosynthesis and accumulation in plant tissues. This uncertainty has in turn limited the potential for manipulation of the synthesis and levels of these important compounds in plants.